The invention relates to the application of matrix substances to surfaces for spatially resolved mass spectrometric measurement of substance distributions in or on these surfaces, especially in histologic thin tissue sections, with ionization by matrix-assisted laser desorption.
The invention provides a method of nebulizing a solution of the matrix substance by vibration without the assistance of gas, and depositing the nebulized droplets, preferably cyclically, on the surface being analyzed.
The state of a tissue section in respect of pathologic change, infection, metabolic anomalies, or stress caused by medication, may be visible in the form of changes to the substance composition compared to the normal state of this tissue. The tissue state can thus be identified from concentration patterns of substances. If the concentrations are sufficiently high, then these concentration patterns can be detected mass spectrometrically. The substances here can be peptides or proteins which are under, or over, expressed and hence form an unusual pattern, but they can also represent posttranslational modifications of proteins, their breakdown products (metabolites), or collections of other substances in the tissue.
Mass spectrometry with ionization of the samples by matrix-assisted laser desorption and ionization (MALDI) has been used successfully for several years for the determination of molecular weights, and for identification and structural characterization of the molecules of substances, particularly of proteins and peptides. In such a case, the protein is usually dissolved and mixed with a solution of a matrix substance such as sinapic acid before being applied to the sample support. The solvent then vaporizes and the matrix substance crystallizes; the protein also crystallizes within the matrix crystals in the form of individual molecules which are widely spaced. If the sample obtained in this way is bombarded with short pulses of laser light with sufficient energy, the matrix substance absorbs energy and explosively vaporizes, the proteins being entrained into the surrounding vacuum of the mass spectrometer by the vapor cloud and ionized by protonation. There are several dozen matrix substances which are suitable in principle; half a dozen different matrix substances have become widely used. Different matrix substances have proved to be optimal for different analytical tasks.
The mass spectrometer separates the ions according to their mass-to-charge ratio (m/z, also termed here the “charge-related mass”) and measures them as a mass spectrum. The mass spectrum can be used to determine their charge-related mass m/z and hence their physical mass m. Since ionization by matrix-assisted laser desorption provides essentially only singly charged ions, we will refer in the following, for the sake of simplification, only to the “mass determination” and not to the determination of the charge-related mass and, correspondingly, simply to the “mass” m of the ions rather than their m/z ratio.
The mass spectrometric analyses can be carried out on individually obtained, homogeneous biological samples such as tissue homogenates, lyzed bacteria and biological fluids (urine, blood serum, lymph, spinal fluid, tears, sputum), the samples generally being subjected to sufficient fractionation beforehand by chromatographic or electrophoretic techniques.
This particularly frees the samples from interfering impurities such as certain buffers, salts or detergents. This removal of interfering impurities of widely varying types is particularly important since they can reduce the ion yield of the MALDI ionization for the analyte substances. The effect of the impurities is not completely understood: it is possible, for example, to observe that simply diluting of the sample, i.e. diluting the impurities as well as the analyte substance in relation to the matrix substance, often brings about a significant improvement of the mass spectrum with respect to the detectability of the analyte substances. The ratio of analyte to impurity remained constant here, indicating that the effect of the impurities is of a higher order than simply linear, and also that there are saturation effects or other suppressive effects for the ionization of the analyte substances. Yet the simple explanation that impurities successfully compete with the analyte substances for the proton sources appears to be incorrect. The presence of detergents, in particular, seems to impede ionization in a general way, without noticeable proportions of ions of these detergents being formed.
The analysis of biological samples thus usually involves very time-consuming sample preparation, particularly if, at the same time, the information concerning the distribution of a protein in different regions of a tissue is to be obtained by measuring individual samples. A method such as “laser capture microdissection” may achieve this but the above-mentioned complex preparation is still necessary, and there is also the difficulty of obtaining sufficient material for this type of analysis.
Imaging mass spectrometry (IMS) makes it unnecessary to go to these lengths. With this method, a thin tissue section having a thickness of between 10 and 20 micrometers is produced, for example using a cryo-microtome, from a frozen piece of tissue taken from an organ of interest, whether human, animal or plant. The thin section is laid on an electrically conductive sample support, for example a conductively coated glass specimen slide. The thin section melts during this process and spreads out smoothly on the sample support. These methods are familiar to the specialist. A layer of a matrix material from a matrix solution is then applied to the dried thin section using a suitable method, which can also involve a reduction in the interfering influence of impurities. After the matrix layer has dried, the specimen slide is introduced directly into the mass spectrometer. There are two different methods for the subsequent mass spectrometric scan: the raster scan method and stigmatic imaging of the ions of a small region.
The raster scan method produces a one- or two-dimensional intensity profile for individual proteins by scanning a thin tissue section with well-focused laser beam pulses in a MALDI mass spectrometer, the proteins being identifiable in the mass spectra obtained for each raster point (U.S. Pat. No. 5,808,300; Caprioli). Each spot is therefore irradiated at least once with a fine-focused pulse of laser light with a diameter of less than 50 micrometers and provides a mass spectrum which can cover a broad range of molecular weights, for example 1 to 30 kilodaltons. Using suitable software, it is then possible to define an ion mass which represents one peptide or one protein, or a narrow mass range around this mass, in the spectra, and to graphically represent its intensity distribution over the surface of the thin tissue section. Using this method it has been possible to correlate the distribution of neuro-peptides in the brain of a rat with specific morphological peculiarities, for example, or to portray the distribution of amyloid beta peptides in the brains of Alzheimer animal models. It is possible to represent spatially precisely defined sections of the brain with “Alzheimer plaques” (Stoeckli M., Staab D., Staufenbiel M., Wiederhold K. H., Signor L., Anal Biochem. 2002, 311, 33-39: Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry).
Stigmatic imaging irradiates a defined area of up to 200 by 200 micrometers with the laser pulse. The ions formed over the area are imaged ion-optically spot by spot onto a spatially resolving detector. It has so far been possible to use this method to scan distribution images of these ion masses by careful selection of individual ion masses (S. L. Luxembourg et al., Anal. Chem. 2003; 75, 1333-41); it is to be expected, however, that very rapid cameras will enable complete mass spectra to be scanned for every spot on the area.
In both cases, raster scan and stigmatic imaging, it is necessary, as described above, to coat the surface of the specimen with a matrix material which absorbs laser energy and ionizes analyte molecules. It is not easy to apply the matrix material in this way because (a) a lateral smearing of the analyte substances must be avoided, (b) the analyte molecules must preferably be extracted from the specimen and embedded into the crystals of the matrix material, and (c) a favorable ratio of analyte molecules to impurities must be achieved. The matrix material here is always applied in dissolved form and crystallizes during a drying process. The solvents are usually mixtures of water and organic solvents, for example acetonitrile. Pure water generally cannot dissolve the matrix substances.
Accordingly, the solvent has to extract analyte molecules from the thin section and transport them vertically into the supernatant solution without distributing them laterally in the process. The organic fractions of the solvent play a particularly important role here, even though many analyte molecules can be dissolved in water. The solvent must first penetrate into the thin layer. The solution on the surface then slowly begins to dry, and consequently the matrix materials crystallize out. The drying process causes the solvents to be drawn out of the thin layer again, a process which probably involves capillary forces and, primarily, osmosis. Analyte molecules are also transported into the drying supernatant solution. It is favorable for the MALDI process if the analyte molecules to be ionized are embedded into the crystals of the matrix material; at the least, the analyte molecules must be in close contact with the matrix materials, for example by being deposited on the grain boundaries of the crystals.
The matrix materials can, for example, be applied by pneumatically spraying a matrix solution in the form of a fine spray droplets onto the thin section as described in U.S. Pat. No. 5,770,272 (Biemann et al.). As described above, the solution must stay on the thin section for a certain length of time before the matrix material crystallizes out in order that the solvent can penetrate into the thin section specimen and the analyte molecules, i.e. mainly the proteins and peptides, can be extracted from the thin section and hence have the chance of being embedded into the crystals. Pneumatic spraying, however, has the disadvantage that it is not possible to prevent the gas stream from macroscopically moving the spray droplets, which have very low viscosity, over the thin section. This reduces the lateral accuracy of the mass spectrometric analysis through lateral smearing.
Pneumatic spraying can also be used for other types of surface whose substance distributions are to be measured. It has been used in this way for thin-layer chromatography (DE 199 37 438 C2; Maier-Posner and Franzen; corresponding to U.S. Pat. No. 6,414,306 B1).
The development of pneumatic spraying for thin section specimens has shown that it is favorable to only spray on a very small amount of matrix solution at a time and to repeat this spraying very frequently. Large quantities of individual spray droplets are applied in each round of spraying but not so many that the spray droplets on the surface can merge to form a film of liquid. Between the individual spraying rounds, the sprayed-on droplets should be able to slowly dry, in around 30 seconds. Thus, for example, the spray is applied for several seconds, followed by a drying period of approx. 30 seconds. A carefully metered gas stream is used for the drying. The spraying processes here typically have to be repeated between a hundred and two hundred times in order to obtain mass spectra which can be readily analyzed. If there are too few spraying cycles, the analyte signals in the mass spectra are noticeably weaker or not present at all.
This method can, of course, be automated, but it is usually carried out manually, generally with so-called airbrush pistols, because of a lack of commercially available instruments. This requires a lot of patience and skill because it takes hours. Even if the user constructs an automatic device himself, the reproducibility is not satisfactory. The gas stream used with pneumatic spraying transports the droplets over macroscopic distances, i.e. up to several millimeters. If the spraying is too “wet”, the droplets merge to form a liquid film in which the gas stream causes the formation of an outward radial flow which delocalizes the analyte molecules. The other extreme is spraying which is too “dry”, where the solvent has already completely vaporized in the flight phase and the matrix impinges on the thin section as a dry crystal shower. In this case, no analyte molecules can be embedded into the matrix. It is very hard to find a happy medium between these two extremes. The two-dimensional spreading of the droplets which are blown onto the surface limits the lateral resolution to around 200 micrometers. The gas stream used for the pneumatic spraying serves to dry the sprayed droplets initially, the organic solvent being the first to escape from the droplets. A larger proportion of water remains, but this is not so good for extracting the proteins from the surface of the thin section. It is therefore necessary to use a large proportion of organic solvent and a low concentration of the matrix material, but this lengthens the process.
The disadvantages of pneumatic spraying have led to the development of another method which does not have this disadvantage of spray droplets in the gas stream: the application of individual droplets by so-called nanospotters. Nanospotting can be done with either piezoelectric or solenoid spotters. Droplets with typical volumes of between 100 picoliters and 10 nanoliters can be shot onto the thin section without contact between nanospotter and section. It has been found that droplets between 10 and 30 micrometers in diameter are most suitable. If the droplets are much smaller, they dry too quickly for the analyte molecules to be efficiently extracted from the tissue. Here too, the liquid must be allowed to act on the thin section for around 30 seconds before the droplets dry, causing the matrix materials to crystallize out completely.
The positioning accuracy of the nanospotter makes it possible to apply the droplets with a lateral repeat precision of the spotter position of a few micrometers, although, depending on the immediate surroundings of the thin section, the droplets are laterally deflected by several micrometers in a statistically distributed direction. It is possible to spot the droplets precisely one on top of the other, but their position does not necessarily correspond to the selected grid. There is a commercial nanospotter on the market for which a method has been developed for applying the droplets in a very precise positioning grid with a separation of 150 to 200 micrometers in each direction. A sufficiently large separation is selected so that the droplets do not run into each other. The droplet size of around 20 micrometers diameter (around ten picograms) results in application spots around 100 micrometers in diameter. Each individual spot is applied again at intervals of around 30 seconds; this application is repeated a few hundred times for each spot. This method of applying the matrix substance also takes several hours.
The mass spectra obtained with this method of nanospotting are high quality but the method is not cheap because a very expensive instrument is used. The method produces much better mass spectra of the analyte substances than is possible with the pneumatic spray method, and a slightly better lateral resolution of around 100 to 150 micrometers, depending on the grid of the spotting. The mass spectrometric measurement requires image recognition to position each individual spot with matrix crystals, however. Since the MALDI process of ionizing analyte molecules can be readily carried out with laser focus points of around 30 micrometers in diameter, this method of nanospotting is not optimally adapted to the achievable lateral resolution of MALDI mass spectrometry.